1. Field of Invention
The present inventio relates to novel natriuretic factors prepared from rat heart atrial muscle which possess diuretic and natriuretic properties of an exceptionally high order together with vasorelaxant and hypotensive properties and anti-hypertensive activities in renovascular hypertensive mammals. Those atrial natriuretic factors which are hereinafter designated as ANFs have the characteristics of peptides composed of from 26-103 amino acid residues are probably involved in the physiological maintenance of water and electrolyte balances in an endocrine role. Furthermore, this invention relates to a process for preparing said ANFs from natural sources and for purifying said native ANFs to substantial homogeneity, as well as to a process for preparing one of said ANFs by synthesis, both in the form of its C-terminal free carboxylic acid and carboxylic acid amide, and to intermediates used in said synthesis. This invention also relates to pharmaceutical preparations containing said native or synthetic ANFs in their free state or as salts with pharmaceutically acceptable acids, and to the use of said pharmaceutical preparations as diuretic and/or natriuretic agents, as vasorelaxants, and as anti-hypertensive agents, for example in mammals suffering from renovascular hypertension.
2. Description of Prior Art
Rat heart atria are known to contain a potent factor of proteinaceous nature able to induce, upon intravenous injection into rats, a strong and immediate diuretic and natriuretic response (de Bold et al., Life Sci., 28, 89-94 (1981)). This substance, called atrial natriuretic factor (ANF), is localized in secretory-like granules of atrial cardiocytes (de Bold, A.J., Can. J. Physiol. Pharmacol., 60, 324-330 (1982); Garcia et al., Experientia, 38, 1071-1073 (1982)).
In recent years, efforts have been devoted to the purification and chemical characterization of ANF in the atrial muscle of the rat and other mammals (Trippodo et al., Proc. Soc. Exp. Biol. Med., 170, 502-508 (1982); Pollock et al., Proc. Fed. Amer. Soc. Exp. Biol., 41, 990A (1982); Kleinert et al., Physiologist, 25, 289A (1982)). However, these studies have been hampered by the low amount of material available from atrial homogenates and also by the apparent heterogeneity of the biologically active factor (Trippodo et al., Proc. Soc. Exp. Biol. Med., 170, 502-508 (1982); Thibault et al., Hypertension, 5 (Suppl. I) 75-80 (1983); Trippodo et al., (1983) Hypertension, 5 (Suppl. I) 81-88; Currie et al., Science, 221, 71-73 (1983)). Initial characterization of the active ANF indicated that it was a basic peptide with a molecular weight of around 5000 (Thibault et al., Hypertension, 5 (Suppl. I) 75-80 (1983); de Bold et al., Life Sci., 33, 297-302 (1983); de Bold, A. J., Proc. Soc. Expt. Biol. Med., 170, 133-138 (1982)). On the other hand, the presence of peptides of higher molecular weight displaying natriuretic activity was also reported (Thibault et al., Hypertension, 5 (Suppl. I) 75-80 (1983); Trippodo et al., (1983) Hypertension, 5 (Suppl. I) 81-88; Currie et al., Science, 221, 71-73 (1983); de Bold et al., Life Sci., 33, 297-302 (1983)). More recently, de Bold et al., (Life Sci., 33, 297-302 (1983)) published the purification by high-pressure liquid chromatography of a low molecular weight peptide with diuretic and natriuretic activities. This peptide, referred to as "cardionatrin-1", was isolated from rat atrial extracts. It possesses a molecular weight of around 5000 and seems to contain 49 amino acids. Recently Flynn and de Bold disclosed the amino acid sequence of cardionatrin-1 in Biochem. Biophys. Res. Comm. Vol. 117, p. 859-865 (1983). Furthermore, Grammer et al., (1983) Biochem. Biophys. Res. Comm. 116, 696-703, have described the isolation from rat heart atria and the purification of a peptide with both vasorelaxant and natriuretic properties and having a molecular weight of approximately 3800. In addition, Thibault et al., (1983) FEBS Letters 164, 286-290, have described the purification of three natriuretic peptides obtained from rat atria, and their respective amino acid compositions seem to indicate that those peptides are composed of 26, 31, and 33 amino acid residues. Finally, Currie et al., (1984) Science 223, 67-69, have described the purification of bioactive atrial peptides (designated as "atriopeptins") and have characterized two of them by determining their respective amino acid sequences which indicate that those atriopeptins are composed of 21 and 23 amino acid residues, respectively.
The process of this invention for preparing native ANF from rat heart atria has the advantage of producing the compounds of this invention in a substantially pure state in reasonably good yields, thus permitting characterization of the compounds by amino acid analysis and unambiguous establishment of their respective constitutions by sequence determinations. Precise knowledge of the constitutions of the compounds of this invention provides the further advantage that the compounds may be prepared by standard methods of peptide synthesis, e.g. as described by Ling et al., "Solid Phase Synthesis of Somatostatin-28", Biochem. Biophys. Res. Comm. 95, 945-951 (1980), the disclosure of which is hereby incorporated herein by reference.
However, in the synthetic process of this invention it has been found to be preferable to combine classical and solid phase procedures, as exemplified below by the preparation of one of the most active compounds by a unique combination of solid-phase and classical methods of peptide synthesis which is applicable to the preparation of all the highly bio-active compounds of this invention. Thus, the synthetic process of this invention has the advantage of providing the peptides of this invention in an economical manner in quantities which are sufficiently large for investigational purposes and for their therapeutic use in the practice of medicine. For example, those peptides may be used to study the mechanisms for maintaining water and electrolyte balances in mammals, or to treat pathological conditions associated with such imbalances. Furthermore, it will be possible to prepare analogs thereof and to use the latter to investigate structure-activity relationships.
Knowledge of the amino acid sequences of these peptides also permits the preparation of the peptides by recombinant DNA techniques. For example, preparation of appropriate DNA sequences by standard techniques and incorporation of these sequences into plasmids. Insertion of the plasmids into bacteria such as E. coli may be followed by fermentation of the bacteria and expression of the peptides. See, for example, Villa-Komaroff et al., A Bacterial Clone Synthetizing Proinsulin, Proc. Natl. Acad. Sci. U.S.A., 75: 3727-3731 (1978), the disclosure of which is hereby incorporated herein by reference.
In the following text the symbols for amino acids are according to the IUPAC--IUB recommendations published in Arch. Biochem. Biophys. 115, 1-12 (1966), except that the following single-letter symbols for the amino acids are also used for the sake of convenience, as shown below together with the conventional symbols:
__________________________________________________________________________ L = Leu = leucine S = Ser = serine S = Asp = aspartic acid A = Ala = alanine C = Cys = cysteine Q = Gln = glutamine G = Gly = glycine F = Phe = phenylalanine N = Asn = asparagine P = Pro = proline I = Ile = isoleucine Y = Tyr = tyrosine R = Arg = arginine K = Lys = lysine W = Trp = tryptophan V = Val = valine E = Glu = glutamic acid T = Thr = threonine __________________________________________________________________________
The symbols for the protective groups used in the synthesis process are described in Schroder and Lubke, "The Peptides", Academic Press, New York and London, 1965, e.g. Boc: t-butyloxycarbonyl; Bzl: benzyl; Acm: acetamidomethyl. Other abbreviations used are e.g. TFA: trifluoroacetic acid; DMF: dimethylformamide; PTH: phenylthiohydantoin; HBT: 1-hydroxybenztriazole; HPLC: high performance liquid chromatography; TLC: thin layer chromatography; other symbols and/or abbreviations are those commonly used in peptide chemistry and will readily be understood by those skilled in the art.
The following terms used in the text of this Application are Registered Trade Marks or Trade Names: C.sub.18 Sep-Pak; Bio-Gel P-10; CM Bio-Gel A; Mono S HR5/5; CN .mu.Bondapak; C.sub.18 .mu.Bondapak; Beckman W3 resin; Dionex DC5A resin; Bio-Sil TSK IEX-530 CM; Polybrene; Quadrol; Sequemat P-6 Autoconverter; Sephadex and Speed Vac.